Abstract

Most organisms use daily light/dark cycles as timing cues to control many essential physiological processes. In plants, growth rates of the embryonic stem (hypocotyl) are maximal at different times of day, depending on external photoperiod and the internal circadian clock. However, the interactions between light signaling, the circadian clock, and growth-promoting hormone pathways in growth control remain poorly understood. At the molecular level, such growth rhythms could be attributed to several different layers of time-specific control such as phasing of transcription, signaling, or protein abundance. To determine the transcriptional component associated with the rhythmic control of growth, we applied temporal analysis of the Arabidopsis thaliana seedling transcriptome under multiple growth conditions and mutant backgrounds using DNA microarrays. We show that a group of plant hormone-associated genes are coexpressed at the time of day when hypocotyl growth rate is maximal. This expression correlates with overrepresentation of a cis-acting element (CACATG) in phytohormone gene promoters, which is sufficient to confer the predicted diurnal and circadian expression patterns in vivo. Using circadian clock and light signaling mutants, we show that both internal coincidence of phytohormone signaling capacity and external coincidence with darkness are required to coordinate wild-type growth. From these data, we argue that the circadian clock indirectly controls growth by permissive gating of light-mediated phytohormone transcript levels to the proper time of day. This temporal integration of hormone pathways allows plants to fine tune phytohormone responses for seasonal and shade-appropriate growth regulation.

Misregulated Genes in Selected Phytohormone Mutants Are Expressed at Dawn under Short-Day Photocycles

(A) The genes that are differentially expressed in the arf6-2arf8-3, abi1-1, and DELLApenta (ga1-3 gai-t6 rga-t2 rgl1-1 rgl2-1) mutants are overrepresented around dawn under short-day photocycles.(B) The genes that are differentially expressed in the ckx1-ox, brx, and ein5-1 mutants are overrepresented around dawn under short-day photocycles.Mutant microarray data from published sources and differentially expressed genes (p < 0.01) were identified by comparing mutant expression to the wild-type expression (). Z-score profile is double plotted for visualization purposes, and the dotted line is the growth rate under short-day photocycles. Hypocotyl growth rate under short-day photocycles (black dotted line) is reproduced to provide a frame of reference for time of day of maximal hypocotyl growth.

(A) The HUD motif (CACATG) is overrepresented in promoter of cycling genes expressed (Z-score, significance score) at midday under continuous light (red) and dawn under short-day photocycles (black) []. The Z-score profile is double plotted for visualization purposes. Black dotted line is the Z-score significance threshold (p < 0.05).(B) Phase overrepresentation of the 54 phytohormone genes with one or more HUD in their promoter (500 bp) under short-day photocycles (black) or continuous light (red).(C) Venn diagram of the 54 phytohormone genes with at least one HUD in their promoter (500 bp) and either the genes that are not growth associated (111 genes, green) or are growth associated (71 genes, blue).(D) Two independent T3 lines (6 and 30) carrying three tandem HUD repeats fused to LUC and a minimal promoter confer night-specific activity under short-day photocycles (black lines) and evening-specific activity under continuous conditions (red lines). Data represent the average of four to six seedlings from two independent experiments. Error bars represent standard error of the mean.

The Growth-Associated Phytohormone Genes Are Linked to Dark-Induced Growth Regulation

To determine the time-dependent behavior of the growth-associated phytohormone genes controlled by the HUD element, the average fold increase between mutant and wild type for each time point is presented (red lines). As a control, the average fold changes for the phytohormone genes not associated with growth that do not contain an HUD element are also presented (black lines). In circadian mutants, phytohormone transcript abundance increases during the dark period compared to wild type, whereas it is constantly higher at any time of the day in phyB mutant.(A) lux-2 versus Ler under intermediate-day photoperiods (12-h light/12-h dark).(B) lhy versus Col under short-day photoperiods (8-h light/16-h dark).(C) phyB-9 versus Col under short-day photoperiods.(D) Short-day versus long-day photocycles.(E) Continuous dark (DD) versus continuous light (LL) [].One-day (six time point) time courses are double plotted for visualization purposes. Black bars represent the dark period. Bars at the bottom of each graph represent light/dark cycle.

(A) Continuous light (black bars) rescues the long hypocotyl defect caused under short-day photocycles (red bars) in lux-2 and lhy mutants, but not in the phyB-9. Plants were grown under continuous light and thermocycles (12-h 22 °C/12-h 12 °C) to ensure synchronization of the circadian clock for expression studies; results without thermocycles (constant 22 °C) were the same as the results presented here and as those described previously [,]. Measurements are an average of at least ten 7-d-old seedlings, and error bars are ± standard deviation. Results represent three independent biological experiments.(B) Continuous light rescues the severe developmental defects of the lhy mutants under short-day photocycles. Two alleles of lhy, lhy (104) and lhy (120), and the parental Ler were grown as in (A) for 2 wk. The long hypocotyl phenotype of lhy under short-day photocycles is completely rescued under continuous light. Results represent three independent biological experiments.(C–E) Continuous light (and thermocycles) as in (A) restores the expression of (C) IAA19, (D) CKX5, and (E) BR6ox2 to wild type in lux-2, but not in phyB-9. The same pattern of expression as in lux-2 was observed for lhy (unpublished data). Expression was measured by qRT-PCR in two independent biological replicates.

The circadian clock and light signaling interact to coordinate a group of different phytohormone transcripts (represented by green, blue, and black lines) to coincide with the dark-to-light transition at dawn. Hypocotyl elongation is a mode of dark growth, so the dark phase of each day is the “growth promotion” period (black box). The circadian clock ensures that growth does not proceed immediately upon exposure to darkness by gating light signaling during the “circadian-maintained light repression” period (red box). The circadian clock maintains light repression during the early evening through light signaling and PHYB activity. In turn, PHYB activity acts indirectly through an unknown gene on at least two cis-acting elements, the HUD and an unknown X element, to repress phytohormone transcript abundance. As night proceeds, circadian maintenance of light signaling decreases, repression of phytohormone transcript abundance is released, and maximal growth occurs at dawn [,]. This model does not attempt to describe hormone abundance or activity. It specifically describes the coordination of phytohormone gene transcript abundance by the circadian clock and light signaling to coincide with the hypocotyl growth window. However, the predictive nature of this model provides a framework for future studies that will directly interrogate the specific interactions of hormones in controlling growth.